April 24, 2013
This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency. The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock increases permeability of the membrane. The results obtained in the experiment show that the E. coli that was transformed with pGLO was able to resist ampicillin and grow in its presence. These results suggest that microorganisms can be genetically engineered to selectively resist certain contaminants, which means that they can potentially be used to human benefit to rid the environment, or even the human body, of unwanted toxins.
Transformation occurs when altered genetic characteristics of bacteria are acquired from a different source. Plasmids are used to transform bacteria because they are small pieces of DNA capable of independently replicating and therefore transferring their (often beneficial) traits to the bacteria. The goal of genetic transformation in this experiment is for the bacteria Escherichia coli to obtain an antibiotic resistance to ampicillin, which can be physically observed when the bacteria expresses the reporter gene Green Fluorescent Protein (GFP) because the transformed bacteria will glow green under UV light when in the presence of arabinose. The gene for GFP is naturally found in a bioluminescent jellyfish, allowing it to glow in the dark. The plasmid used to transform the bacteria contains the antibiotic resistance along with a gene that codes for the fluorescent protein.
The objective of this experiment is to genetically engineer ampicillin resistant E. coli by transforming it with a plasmid containing an antibiotic resistance gene and a gene that codes for GFP and to calculate the transformation efficiency. This will alter the phenotype by inserting foreign DNA. This experiment is important because it demonstrates a technique that has practical applications in areas such as environmental concern. Bacteria that have been genetically altered can obtain traits that allow it to be used to break down toxic compounds (i.e. oil spill) to reduce the amount of harmful substances found in soil and water (Spilios, 2013). Some environmental contaminants are actually toxic for microorganisms, like bacteria, and deactivate the cells abolishing their ability to break down the contaminants (Pieper, 2000). For this reason, bacteria that are resistant to specific toxins are preferable and it would be desirable to replicate their DNA– the resistance gene specifically –and use it to create such resistance in other organisms. This research builds directly from the knowledge used in this experiment transforming E. coli.
The hypotheses for this experiment are that the bacteria will be transformed by the plasmid to develop an antibiotic resistance and will grow despite the presence of ampicillin. Additionally, when arabinose sugar is added to the strain of bacteria transformed by the plasmid, it will express GFP and glow green under UV light.
The methods and procedures for transforming bacteria in this experiment were performed according to Spilios (2013). Bacteria strains of E. coli were genetically transformed with a plasmid to carry ampicillin resistance. 250 microliters of a CaCl2 transformation solution were pipetted into closed test tubes, which were then placed on ice. A starter plate with E. coli colonies was observed and two colonies were transferred into the tubes of CaCl2, one colony in each, and dispersed evenly throughout the solution by mixing. The tubes were then placed back on ice. Ten nanograms of pGLO DNA solution was then added to one of the tubes and the tubes were incubated on
ice for ten minutes. While the tubes were incubating, four plates of LB nutrient agar were obtained, one containing just LB, two also with ampicillin, and one also with ampicillin and arabinose. The test tubes after the ten minutes were incubated for 50 seconds in a 42 degree Celsius water bath and then returned to the ice for another two minutes. The tubes were then removed from the ice and 250 microliters of LB nutrient broth was added to each tube, which was then incubated for an additional ten minutes at room temperature. 100 microliters of the solution in the tube containing pGLO was added to one plate with just LB agar and ampicillin and to the plate with ampicillin and arabinose. 100 microliters of the solution in the tube without pGLO was added to the other plate containing LB and ampicillin as well as to the plate containing only LB. The solution was spread around the surface of each agar using a new sterile loop for each plate. The plates were then incubated at 37 degrees Celsius and results were observed and recorded after 27 hours.
Yellow colonies; still yellow under UV light
Yellow colonies; glow green under UV light
Table 1. Transformed Plates After Incubation shows that there was growth of transformed bacteria and their observable characteristics.
Yellow colonies; still yellow under UV light
Table 2. Control Plates After Incubation shows whether there was growth of E. coli without the presence of the pGLO plasmid after exposure to ampicillin.
The transformed bacteria showed growth despite the presence of ampicillin (Table 1), whereas the control plate with ampicillin did not show any growth, and the control plate with only LB agar showed the formation of a lawn of bacteria (Table 2). The transformed bacteria on the plate with LB, ampicillin and arabinose differed from the transformed plate without arabinose in that they glowed green under UV light. The bacteria without arabinose maintained an unaltered appearance under UV light. The transformation efficiency for the transformed bacteria was 5.2 × 104 transformants per microgram of DNA.
In this experiment the objective was to transform E. coli with the pGLO plasmid and calculate the transformation efficiency. The hypotheses were that the plate with only LB agar and untransformed E. coli would grow a lawn; the control plate of untransformed bacteria with LB and ampicillin would experience no growth; the transformed plate with just LB and ampicillin would grow colonies of bacteria but it would not glow green under UV light; and the transformed plate with LB, ampicillin and arabinose would grow colonies that would glow green under UV light. The results found supported each of these hypotheses as the bacteria grew as predicted. The untransformed bacteria grew on the LB plate rapidly as none of them were inhibited by the ampicillin, but they did not grow when exposed to ampicillin on the other control plate because it did not have the gene for antibiotic resistance. The transformed plates both carried the gene to resist ampicillin from the pGLO plasmid, so they were able to grow colonies that were the offspring of the genetically transformed E. coli. The plate that also contained arabinose grew colonies of bacteria that contained the gene that not only resists ampicillin but also expresses GFP and causes the bacteria to glow green under UV light. The arabinose operon in E. coli is a
set of genes that will only become active when the sugar arabinose is present (Spilios, 2013). Genes are transcribed and translated to break down arabinose for energy. The expression of enzymes that break down arabinose can be turned on or off. In the pGLO plasmid, the GFP gene has replaced genes that code for the break down of arabinose. In this experiment, the arabinose added acted as the “on switch” to cause the operon to produce GFP. The transformation efficiency calculated for the results was 5.2 × 104 transformed cells per microgram of DNA used. This is slightly shy of the standard transformation efficiency in standard published research. Hanahan’s (1983) experiment states that he improved on the levels of efficiency observed at standard conditions by 100- to 1000-fold with reported efficiency of 5 × 108 transformants per microgram of plasmid. The type growth or nutrient media does not make a large impact on the transformation efficiency, but the concentrations on components that increase growth rate in the media tend to aid in transformation (Hanahan, 1983). Therefore, it may prove beneficial to incorporate a growth medium with higher concentrations of Mg2+ into the experimental design to achieve slightly higher transformation efficiency. Future experiments may focus on working to increase the transformation efficiency of E. coli (Chung, 1989). This can help decrease costs and increase reliability to replace existing methods. As previously stated, developing a working knowledge and understanding of genetic engineering aids in addressing environmental and health concerns. Microorganisms could be used to target specific contaminants or toxins in the environment or in the human body.
Chung CT. 1989. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences of the United States of America, 86:2172-2175.
Hanahan D. 1983. Studies on Transformation of Escherichia coli with Plasmids. Journal of Molecular Biology, 166:557-580.
Pieper DH. 2000. Engineering bacteria for bioremediation. Current Opinion in
Spilios K (ed). 2013. Principles of Biology, II. Hayden-McNeil Publishing, Plymouth, MI. Module #6, pp 119-127.